Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin

Miyagawa, Daichi; Makino, Yasushi; Sato, Masaaki

doi:10.1093/jb/mvv097

Cited by 3 publications

(3 citation statements)

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“…To perform the GP ab study correctly and consistently, we modified previously reported procedures for the preparation and identification of GP ab : First, PhK-catalyzed phosphorylation of rabbit muscle GP b was performed according to our previously reported method (Miyagawa et al 2016 ), except that gelatin (protein adsorption-preventing agent) and ethylenediaminetetraacetic acid (EDTA; PhK inactivator) were not added to prevent disruption of the subsequent HPLC purification. Instead, the reaction product was immediately subjected to DEAE-5PW anion-exchange HPLC.…”

Section: Resultsmentioning

confidence: 99%

“…Muscle GP is allosterically activated by AMP (Lowry et al 1964 ; Madsen et al 1983 ; Rush and Spriet 2001 ). Notably, muscle GP a exhibits a very strong affinity for AMP ( K d , ~ 0.3 μM) compared with muscle GP b ( K d , ~ 300 μM) (Miyagawa et al 2016 ). As previously stated, Burkhardt and Wegener ( 1994 ) reported that GP ab from hawk moth muscle showed a single and unique AMP dependence ( K d , ~ 30 μM), suggesting that phosphorylation of either subunit of the GP dimer partially, but not fully, changed the AMP-binding site structures of both subunits.…”

Section: Resultsmentioning

confidence: 99%

“…A mixture (300 μL) containing 50 mM Tris–HCl buffer (pH 8.2), 3.0 mM ATP, 10 mM MgCl 2 , 0.30 mM CaCl 2 , 40 mM sodium fluoride, 3.0 mM 2-mercaptoethanol, 0.30 mg rabbit muscle GP b , and 2.0 U rabbit muscle PhK was incubated at 37 °C for 10 min (Miyagawa et al 2016 ). One unit of PhK was defined as the amount of enzyme that produces 1.0 μg of GP a per minute under the employed conditions.…”

Section: Methodsmentioning

confidence: 99%

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Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites

Kamada,

Ikeda,

Makino

et al. 2024

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Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites

Kamada,

Ikeda,

Makino

et al. 2024

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Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives

et al. 2017

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Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG, SG, SG, SG, SG, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc -AltNAc-Glc -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG -SG subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V ) with that toward PA-0 (V), we found that the value of V /V decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.

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A new interpretation of sulfate activation of rabbit muscle glycogen phosphorylase

2018

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It is widely known that sulfate ion at high concentration serves like an allosteric activator of glycogen phosphorylase (GP). Based on the crystallographic studies on GP, it has been assumed that the sulfate ion is bound close to the phosphorylatable Ser site of nonactivated GP, causing a conformational change to catalytically-active GP. However, there are also reports that sulfate ion inhibits allosterically-activated GP by preventing the phosphate substrate from attaching to the catalytic site. In the present study, using a high concentration of sulfate ion, significant enhancement of GP activity was observed when macromolecular glycogen was used as substrate but not when smaller maltohexaose was used. In glycogen solution, nonreducing-end glucose residues are localized on the surface of glycogen and are not distributed homogenously in the solution. Using cyclodextrin-immobilized column chromatography, we found that sulfate at high concentration promoted GP-dextrin binding through the dextrin-binding site (DBS) located away from the catalytic site. This result is consistent with the properties of the DBSs found in glycogen-debranching enzyme and β-amylase. Therefore, we propose a new interpretation of the sulfate activation of GP, wherein sulfate ions at high concentration promote glycogen-binding to the DBS directly, and glycogen-binding to the catalytic site indirectly. Our findings were successfully applied to the affinity purification of porcine brain GP.

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Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin

Cited by 3 publications

References 53 publications

Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites

Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives

A new interpretation of sulfate activation of rabbit muscle glycogen phosphorylase

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